Genomic instability in long-term culture of human adipose-derived mesenchymal stromal cells.

Mesenchymal stromal/stem cells stem (MSC) have been widely studied due to their great potential for application in tissue engineering and regenerative and translational medicine. In MSC-based therapy for human diseases, cell proliferation is required to obtain a large and adequate number of cells to ensure therapeutic efficacy. During in vitro culture, cells are under an artificial environment and manipulative stress that can affect genetic stability. Several regulatory agencies have established guidelines to ensure greater safety in cell-based regenerative and translational medicine, but there is no specific definition about the maximum number of passages that ensure the lowest possible risk in MSC-based regenerative medicine. In this context, the aim of this study was to analyze DNA damage and chromosome alterations in adipose-derived mesenchymal stromal cells (ADMSC) until the eleventh passage and to provide additional subsidies to regulatory agencies related to number of passages in these cells. Thus, two methods in genetic toxicology were adopted: comet assay and micronucleus test. The comet assay results showed an increase in DNA damage from the fifth passage onwards. The micronucleus test showed a statistically significant increase of micronucleus from the seventh passage onwards, indicating a possible mutagenic effect associated with the increase in the number of passages. Based on these results, it is important to emphasize the need to assess genetic toxicology and inclusion of new guidelines by regulatory agencies to guarantee the safety of MSC-based therapies for human diseases.

Genomic instability in long-term culture of human adipose-derived mesenchymal stromal cells

Mesenchymal stromal/stem cells stem (MSC) have been widely studied due to their great potential for application in tissue engineering and regenerative and translational medicine. In MSC-based therapy for human diseases, cell proliferation is required to obtain a large and adequate number of cells to ensure therapeutic efficacy. During in vitro culture, cells are under an artificial environment and manipulative stress that can affect genetic stability. Several regulatory agencies have established guidelines to ensure greater safety in cell-based regenerative and translational medicine, but there is no specific definition about the maximum number of passages that ensure the lowest possible risk in MSC-based regenerative medicine. In this context, the aim of this study was to analyze DNA damage and chromosome alterations in adipose-derived mesenchymal stromal cells (ADMSC) until the eleventh passage and to provide additional subsidies to regulatory agencies related to number of passages in these cells. Thus, two methods in genetic toxicology were adopted: comet assay and micronucleus test. The comet assay results showed an increase in DNA damage from the fifth passage onwards. The micronucleus test showed a statistically significant increase of micronucleus from the seventh passage onwards, indicating a possible mutagenic effect associated with the increase in the number of passages. Based on these results, it is important to emphasize the need to assess genetic toxicology and inclusion of new guidelines by regulatory agencies to guarantee the safety of MSC-based therapies for human diseases.

Investigation of taxol as a potential radiation sensitizer.

BACKGROUND: The authors evaluated the effects of taxol, a microtubular inhibitor, as a possible radiation sensitizer on the human leukemic cell line (HL-60). Taxol acts as a mitotic inhibitor, blocking cells in the G2M-phase of the cell cycle. The differential radiation sensitivity of cells in various phases of the cell cycle has been well recognized. This study was focused on the possible interaction between radiation and a microtubular inhibitor, taxol, in regard to its ability to synchronize cells at the G2M-phase of cell cycle and, thereby, enhance the radiation sensitivity of the cells. METHODS: HL-60 cells were exposed to 3 x 10(-8) M concentrations of taxol for 1 hour at 37 degrees C followed by reculturing for 24 hours in drug-free medium. The cells were then seeded into 60-mm diameter plastic dishes at appropriate cell concentrations to estimate their colony-forming efficiency. The radiation dose ranged from 0-400 cGy and was delivered in a single fraction. The cellular survival after treatment with the drug and/or radiation was determined using a soft agar clonogenic assay. RESULTS: When HL-60 cells were treated with taxol, up to 70% of the cells were blocked in G2M-phase, as determined by flow cytometric analysis. At the low cytotoxic dose of 3 X 10(-8) M, the sensitizing enhancement ratio was 1.48. CONCLUSIONS: It appears that taxol has a radiation-sensitizing effect on HL-60 cells and deserves further investigation with other cell lines.